Role of 15-hydroxyeicosatetraenoic acid in hemozoin-induced lysozyme release from human adherent monocytes

Natural Haemozoin Induces Expression and Release ofHuman Monocyte Tissue Inhibitor of Metalloproteinase-1Manuela Polimeni1, Elena Valente1, Daniela Ulliers1, Ghislain Opdenakker2, Philippe E. Van den Steen2,

Giuliana Giribaldi1., Mauro Prato1,3*.

1Dipartimento di Oncologia, Universita di Torino, Torino, Italy, 2 Laboratory of Immunobiology, Rega Institute, Catholic University of Leuven, Leuven, Belgium,

3Dipartimento di Neuroscienze, Universita di Torino, Torino, Italy

Abstract

Recently matrix metalloproteinase-9 (MMP-9) and its endogenous inhibitor (tissue inhibitor of metalloproteinase-1, TIMP-1)have been implicated in complicated malaria. In vivo, mice with cerebral malaria (CM) display high levels of both MMP-9 andTIMP-1, and in human patients TIMP-1 serum levels directly correlate with disease severity. In vitro, natural haemozoin (nHZ,malarial pigment) enhances monocyte MMP-9 expression and release. The present study analyses the effects of nHZ onTIMP-1 regulation in human adherent monocytes. nHZ induced TIMP-1 mRNA expression and protein release, andpromoted TNF-a, IL-1b, and MIP-1a/CCL3 production. Blocking antibodies or recombinant cytokines abrogated or mimickednHZ effects on TIMP-1, respectively. p38 MAPK and NF-kB inhibitors blocked all nHZ effects on TIMP-1 and pro-inflammatorymolecules. Still, total gelatinolytic activity was enhanced by nHZ despite TIMP-1 induction. Collectively, these data indicatethat nHZ induces inflammation-mediated expression and release of human monocyte TIMP-1 through p38 MAPK- and NF-kB-dependent mechanisms. However, TIMP-1 induction is not sufficient to counterbalance nHZ-dependent MMP-9enhancement. Future investigation on proteinase-independent functions of TIMP-1 (i.e. cell survival promotion and growth/differentiation inhibition) is needed to clarify the role of TIMP-1 in malaria pathogenesis.

Citation: Polimeni M, Valente E, Ulliers D, Opdenakker G, Van den Steen PE, et al. (2013) Natural Haemozoin Induces Expression and Release of Human MonocyteTissue Inhibitor of Metalloproteinase-1. PLoS ONE 8(8): e71468. doi:10.1371/journal.pone.0071468

Editor: Emma H. Wilson, University of California, Riverside, United States of America

Received January 29, 2013; Accepted June 29, 2013; Published August 14, 2013

Copyright: � 2013 Polimeni et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was financed by intramural funds from Universita di Torino to G. Giribaldi, by funds from Mrs. Franca Squazza and from Societa Italiana diBiochimica (SIB) to M. Prato, and by Fund for Scientific Research (FWO-Vlaanderen) and Research Fund of the KULeuven (GOA/2012/017) to G. Opdenakker and P.Van den Steen. M. Prato holds a professorship granted by Universita di Torino and Azienda Sanitaria Locale-19 (ASL-19). P. Van den Steen holds a researchprofessorship of the KULeuven. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

. These authors contributed equally to this work.

Introduction

As a consequence of the global eradication program launched

by charity foundations [1], World Health Organization (WHO)

officially registered in 2010 a decline in estimated malaria cases

and deaths, with 655,000 deaths counted among more than 200

million clinical cases worldwide, of which 91% were due to

Plasmodium falciparum [2]. Nevertheless, malaria remains an

alarming emergency in developing countries, with the vast

majority of cases occurring in the African Region (81%) and

South-East Asia (13%) [2]. Thus it is imperative to investigate new

anti-malarial drugs for primary and adjuvant therapy [3] and

identify new affordable markers for early diagnosis of malaria.

Human matrix metalloproteinases (MMPs) are a family of

proteolytic enzymes involved in wide variety of biological functions

including modulation of inflammatory response, disruption of

inter-endothelial tight junctions, and degradation of sub-endothe-

lial basal lamina [4–7]. As such, they are good candidate molecules

and indeed there is growing evidence that MMPs play critical roles

in malaria in both animal and human disease models (see [8–10]

for more extensive reviews). Notably, malarial pigment (nHZ,

natural haemozoin), a waste product of haemoglobin digestion by

Plasmodium parasites, induces MMP-9 release from human

monocytes [11–14] and endothelial cells [9,15–16], and synthetic

HZ (sHZ) interacts with proMMP-9 priming its activation by

MMP-3 [17].

Endogenous inhibitors of MMPs (TIMPs, tissue inhibitors of

metalloproteinases) represent one mode of MMP regulation [18];

however, their involvement in malaria has been scarcely investi-

gated, and their role remains debated. A few lines of evidence from

animal and human models support the involvement of TIMPs in

malaria. CM-sensitive mice infected with P. berghei ANKA display

increased mRNA expression of TIMP-1 in the brain, and both

TIMP-1 and -3 mRNA is increased in the liver and spleen, whilst

mRNA levels of TIMP-2 and -4 remain unchanged [19].

Increased serum levels of TIMP-1 are also found in Rhesus

macaques (Macaca mulatta) experimentally infected with P. coatneyi,

a simian malaria parasite that closely mimics the biological

characteristics of P. falciparum and replicates the multisystemic

dysfunction of human severe malaria [20]. Human patients with

severe or uncomplicated malaria have higher serum TIMP-1 levels

compared to healthy controls suggesting TIMP-1 may be a

valuable marker of disease severity [21]. However, the cellular

source of TIMP-1 and the mechanisms underlying TIMP-1

enhancement are as of yet unidentified. Additionally, it is

imperative to assess whether increased CM-associated TIMP-1

levels are sufficient to counterbalance nHZ-enhanced MMP-9.

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Endothelial cells and monocytes are both producers of inducible

TIMP-1 protein [22–23], and their phenotype and functions can

be directly affected by malarial products such as infected red blood

cells (IRBCs) and nHZ [9]. However, it is unlikely that

endothelium is the TIMP-1 source in malaria, as neither IRBCs

nor nHZ affect TIMP-1 protein release from human microvas-

cular endothelial cells [15–16].

Here we investigate the in vitro effects of nHZ on human

monocyte TIMP-1 gene expression and protein release, the

responsible soluble mediators, the signaling pathways involved,

and the net gelatinolytic activity resulting from the altered MMP-

9/TIMP-1 balance.

Materials and Methods

MaterialsAll materials were from Sigma-Aldrich, St Louis, MO, unless

otherwise stated. Sterile plastics were from Costar, Cambridge,

UK; Percoll was from Pharmacia, Uppsala, Sweden; Diff-Quik

parasite stain was from Baxter Dade AG, Dudingen, Switzerland;

Panserin 601 monocyte medium was from PAN Biotech,

Aidenbach, Germany; ELISA kits for hTNF-a and hIL-1b assays

were from Cayman, Ann Arbor, MI; blocking anti-hTNF-a/IL-1bantibodies and rhTNF-a/IL-1b were from Merck, Darmstadt,

Germany; ELISA kit for hMIP-1a/CCL3, anti-hMIP-1a/CCL3

blocking antibodies and rhMIP-1a/CCL3 were from R&D

Systems, Minneapolis, MN; p38 MAPK inhibitor SB203580 was

from Cell Signaling Technology, Danvers, MA; ELISA kits for

hMMP-9 and hTIMP-1 were from RayBiotech, Norcross, GA;

cell culture medium RPMI, DQ-gelatin, TRIzol, M-MLV, oligo-

dT, sense and anti-sense primers, Platinum Taq DNA Polymerase

were from Invitrogen, Carlsbad, CA; DNA-free kit was from

Ambion, Austin, TX; Beacon Designer 7.0 software was from

Premier Biosoft International, Palo Alto, CA; dNTPs were from

Applied Biosystem, Foster City, CA; anti-hTIMP-1 (sc-21734)

monoclonal antibodies were from Santa Cruz Biotechnology,

Santa Cruz, CA; iCycler iQ Real Time Detection System

Software version 3.0 and electrophoresis reagents were from

Bio-Rad Laboratories, Hercules, CA; computerized densitometer

Chemidoc was from Biorad, Hercules, CA; Synergy HT

microplate reader was from Bio-Tek Instruments, Winooski, VT;

recombinant proMMP-9 and MMP-9 were produced as previ-

ously described [24].

Cultivation of Plasmodium falciparum and Isolation ofnHZPlasmodium falciparum parasites (Palo Alto strain, Mycoplasma-

free, LPS-free) were kept in culture as described [25]. After

centrifugation at 5,000g on a discontinuous Percoll-mannitol

density gradient, nHZ was collected from the 0–40% interphase.

nHZ was washed five times with 10 mM HEPES (pH 8.0)

containing 10 mM mannitol at 4uC and once with phosphate-

buffered saline (PBS). nHZ was treated with DNase to remove any

adhering nuclear material as previously described [26]. nHZ was

stored at 20% (v/v) in PBS at 220uC or immediately used for

opsonization and phagocytosis.

Preparation and Handling of MonocytesHuman monocytes were separated by Ficoll centrifugation [11]

from freshly collected buffy coats discarded from blood donations

by healthy adult donors of both sexes provided by the local blood

bank (AVIS, Associazione Volontari Italiani Sangue, Torino,

Italy). Separated lympho/monocytes were resuspended in RPMI

medium and plated on six-well plates. Each well received 86106

cells. The plates were incubated in a humidified CO2/air-

incubator at 37uC for 60 min. Thereafter, non-adherent cells

were removed by three washes with RPMI and remaining

adherent cells (,16106 monocytes/well) were again incubated

at 37uC overnight. Shortly before starting phagocytosis, wells were

washed with RPMI and Panserin 601 monocyte medium was

added (2 ml/well).

Pre-selection of NF-kB-non-activated Monocytes by FACSAnalysis and Real Time RT-PCR

Before starting experiments, a pre-selection of cell populations

was taken as a precautionary measure, as previously described

[13]. Briefly, cell cultures isolated through Ficoll separation were

analyzed by flow cytometry. Only cell populations showing at least

70% monocytes were used for following experiments. Additionally,

in order to avoid the use of NF-kB pre-activated monocytes, cells

were analyzed by Real Time RT-PCR: in each cell preparation a

cell aliquot was stimulated or not with LPS (1 mg/ml) for 4 h, and

TNF-a RNA production measured in lysates by Real Time RT-

PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was

used as housekeeping gene. Only unstimulated monocyte popu-

lations (NF-kB-non-activated cells) showing at least a 3-PCR-

cycles gap of cDNA amplification between controls and LPS-

stimulated cells were used for the subsequent experiments.

Cell Culturing Experimental Conditions: Phagocytosis ofnHZ or Latex Particles and Treatment with BlockingAntibodies, Recombinant Pro-inflammatory Molecules, orCell Signalling Chemical Inhibitors

Phagocytosis assay was performed as previously described [27].

Briefly, nHZ (120 nmoles HZ haem, an amount comparable to

50 ml trophozoites on haem content basis) and 50 ml amine-

modified latex particles (2.5% solids, diameter 0.105 mm) were

added to each well of a six-well plate containing the same amounts

of human adherent monocytes (,16106 cells/well). nHZ and

latex particles were opsonized with fresh autologous serum. After

opsonization, all phagocytic meals were suspended in Panserin 601

monocyte medium. The plates were centrifuged at low speed for

5 s to start phagocytosis and incubated in a humidified CO2/air-

incubator at 37uC for 2 h - a time period maximizing phagocytosis

but not sufficient to induce haem-oxygenase-mediated degradation

of ingested haem [28]. Cells were checked by optical microscopy:

as an average, nHZ-containing monocytes were 25–35% among

the total cells, a percentage similar to in vivo levels measured in

patients with severe malaria showing high parasitaemia [29].

Additionally, the amount of nHZ phagocytosed by monocytes was

quantified by luminescence: as an average, each monocyte

ingested nHZ equivalent to ,8–10 trophozoites in term of

ingested haem, in line with previous results from our group [30].

Thereafter, non-ingested nHZ and latex particles were removed

by four washes with RPMI. The plates were then incubated in

Panserin 601 medium in a humidified CO2/air-incubator at 37uCfor the indicated times.

In selected experiments, unfed and nHZ-fed monocytes were

incubated for the indicated times with 30 ng/ml anti-hTNF-a,

anti-hIL-1b or anti-hMIP-1a/CCL3 blocking antibodies; 20 ng/

ml rhTNF-a, rhIL-1b or rhMIP-1a/CCL3; 10 mM SB203580,

15 mM quercetin, 10 mM artemisinin, or 10 mM parthenolide,

dissolved in DMSO (final solvent concentration less than 0.1%

v/v).

Complimentary co-culturing experiments with unfed and nHZ-

fed monocytes were also performed using six-well plate Transwell

systems with 0.4 mm of porosity (see Figure S2A-B). Briefly,

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monocytes (0,56106 cells/transwell) were seeded in transwells

(provisionally placed in cell-free wells) and fed with nHZ for 2h.

Thereafter, transwells with nHZ-fed monocytes were moved into

new wells containing unfed monocytes (16106 cells/well) and

incubated for 2 h, a time previously shown to be sufficient to

induce early production of TNF-a, IL-1b, MIP-1a/CCL3 from

nHZ-fed cells [25,31]. As negative and positive controls, respec-

tively, unfed monocytes were also left unfed or fed with nHZ for

2 h. Then nHZ and transwells with nHZ-fed monocytes were

removed. After washing and adding fresh medium, cells were

incubated in Panserin 601 medium in a humidified CO2/air-

incubator for 24 h before collecting supernatants for subsequent

analyses.

Cytotoxicity Studies by Lactate Dehydrogenase AssayThe potential cytotoxic effects of phagocytic meals (nHZ and

latex) and treatments (anti-hTNF-a, anti-hIL-1b and anti-hMIP-

1a/CCL3 antibodies; rhTNF-a, rhIL-1b and rhMIP-1a/CCL3;

SB203580, quercetin, artemisinin, and parthenolide) were mea-

sured as the release of lactate dehydrogenase (LDH) from cells into

the extracellular medium (see Figure S1A–B).

Briefly, human adherent monocytes were left unfed or fed with

phagocytic meals for 2 h; after washings, unfed and fed cells were

incubated for 24 h in the presence or in the absence of each one of

the above mentioned treatments. Then, 1 ml of cell supernatants

was collected and centrifuged at 13000 g for 2 min. Cells were

washed with fresh medium, detached with trypsin/ethylenedi-

aminetetraacetic acid (EDTA) (0.05/0.02% v/v), washed with

PBS, resuspended in 1 ml of TRAP (82.3 mM triethanolamine,

pH 7.6), and sonicated on ice with a 10 s burst. 5 ml of cell lysates

and 50 ml of cell supernatants were diluted with TRAP and

supplemented with 0.5 mM sodium pyruvate and 0.25 mM

NADH (300 mL as a final volume) to start the reaction. The

reaction was followed measuring the absorbance at 340 nm (37uC)

with a microplate reader. Both intracellular and extracellular

enzyme activities were expressed as mmol of oxidized NADH/

min/well. Finally, cytotoxicity was calculated as the net ratio

between extracellular and total (intracellular+extracellular) LDH

activities. Cell viability was calculated as the net ratio between

intracellular and total LDH activities.

Measurement of TIMP-1 mRNA Levels by Real Time RT-PCR

After the end of phagocytosis, monocytes (16106 cells/well)

were further incubated with Panserin 601 monocyte medium in a

humidified CO2/air-incubator at 37uC for 15 h. Total cellular

RNA from 26106 cells (contained in 2 wells) was isolated from

monocytes by TRIzol, according to the manufacturer’s instruc-

tions, and eluted in 20 ml diethyl pyrocarbonate water. To remove

any contaminating DNA, RNA was treated with Ambion’s DNA-

free kit. Subsequently, 6 mg of RNA were reverse transcribed into

single-stranded cDNA using M-MLV (200 U/ml final concentra-

tion) and oligo-dT (25 mg/ml final concentration). Real Time RT-

PCR analysis was performed with the iCycler Instrument and the

iCycler iQ Real Time Detection System Software version 3.0 as

previously described [11]. For TIMP-1 (GenBank accession no.

BC000866) primers (forward: 59-AGA CGG CCT TCT GCA

ATT CC-39, reverse: 59-GCT GGT ATA AGG TGG TCT GGT

T-39), oligonucleotide sequences were identified using Beacon

Designer Software package and designed to be intron-spanning

allowing the differentiation between cDNA and genomic DNA-

derived PCR products. GAPDH (GenBank accession no.

BC020308) was used as housekeeping gene; primer sequences

were from the Bio-Rad library: forward: 59-GAA GGT GAA

GGT CGG AGT-39 and reverse: 59-CAT GGG TGG AAT CAT

ATT GGA A-39. For each 25 ml PCR reaction mix: 1 ml cDNA

(corresponding to 105 cells); 1.0 ml sense primer (10 mM); 1.0 ml

anti-sense primer (10 mM); 0.5 ml dNTP (10 mM); 1.5 ml MgCl2(50 mM); 1.25 U Platinum Taq DNA Polymerase; 2.5 ml Buffer

(10x); 1.7 ml SYBR Green (stock 1:10,000); and 14.55 ml PCR-

grade water were mixed together. DNA polymerase was pre-

activated for 2 min at 94uC. TIMP-1 amplification was performed

by 50 cycles with denaturation at 54uC for 30 s, annealing at 57uCfor 40 s and extension at 72uC for 30 s; GAPDH amplification

was performed by 40 cycles with denaturation at 94uC for 30 s,

annealing at 65uC for 30 s and extension at 72uC for 30 s for

GAPDH. Relative quantification for TIMP-1, expressed as fold

variation over untreated control cells, was calculated with the

efficiency-corrected quantification model [32] after determining

the difference between CT of the given gene A (TIMP-1) and that

of the calibrator gene B (GAPDH). CT values are means of

triplicate measurements. To validate the use of the method we

tested serial dilutions of cDNA from monocytes, stimulated for

15 h by 20 ng/ml rhTNF-a. The specificity of PCR was

confirmed by melt curve analysis. The melting temperatures for

each amplification product were 87.4uC for TIMP-1 and 86.5uCfor GAPDH.

Assay of TIMP-1 Protein Levels by Western BlottingAfter the end of phagocytosis, monocytes (16106 cells/well)

were further incubated with Panserin 601 monocyte medium in a

humidified CO2/air-incubator at 37uC for 24 h. Thereafter, 15 ml

of cell supernatants were separated on a 12% polyacrylamide

denaturing gel, transferred to a polyvinylidene difluoride mem-

brane, and probed with monoclonal anti-TIMP-1 antibodies.

After staining with secondary anti-mouse HRP-conjugated anti-

bodies, bands were visualized by ECL staining. Densitometric

analysis of the bands considered to reflect total protein levels was

performed using a computerized densitometer with protein levels

presented in relative units compared to background.

Measurement of TNF-a, IL-1b, MIP-1a/CCL3, MMP-9 andTIMP-1 Production by ELISA

After the end of phagocytosis, monocytes (16106 cells/well)

were further incubated with Panserin 601 monocyte medium in a

humidified CO2/air-incubator at 37uC up to 24 h. Cell superna-

tants were collected 6 h, 15 h and 24 h after phagocytosis. The

levels of soluble TNF-a, IL-1b, MIP-1a/CCL3, MMP-9 and

TIMP-1 were assayed in 100 ml of monocyte supernatants by

specific ELISA. Standard calibration curves were generated with

rhTNF, rhIL-1b, rhMIP-1a/CCL3, rhMMP-9 and rhTIMP-1

according to the manufacturer’s instructions.

Assay of Total Gelatinolytic Activity by a FluorogenicGelatin Conversion Assay

After phagocytosis, monocytes (16106 cells/well) were further

incubated with Panserin 601 monocyte medium in a humidified

CO2/air-incubator at 37uC for 24 h. Thereafter, the total

gelatinolytic activity [the net overall activity of gelatinases

(MMP-2 and MMP-9) and their inhibitors (TIMP-2 and TIMP-

1, respectively)], was measured in cultured supernatants using DQ-

gelatin as recently described [33]. Briefly, DQ-gelatin was

dissolved at a stock concentration of 1 mg/ml in pure water.

Serial dilutions of the samples were prepared in a black microtiter

plate, and DQ-gelatin was added at a final dilution of 2.5 mg/ml in

50 mM Tris/HCl pH 7.6, 150 mM NaCl, 5 mM CaCl2, 0.01%

Tween-20. Immediately thereafter, the plate was placed in the

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fluorescence reader and fluorescence measured every 10 min for

2 h at 37uC (ex. 485 nm/em. 530 nm). As a standard, serial

dilutions of recombinant MMP-9 activated by MMP-3 were

included in each plate. The results were expressed as gelatinolytic

activity units per ml, with 1 unit corresponding to 37 ng/ml

activated rhMMP-9.

Statistical AnalysisFor each set of experiments, data are shown as means+SEM

(Real Time RT-PCR, densitometry, ELISA and fluorogenic

gelatin conversion assay) or as a representative image (Western

blotting) of three independent experiments with similar results. All

data were analyzed by a one-way Analysis of Variance (ANOVA)

followed by Tukey’s post-hoc test (software: SPSS 16.0 for

Windows, SPSS Inc., Chicago, IL).

Results

nHZ Induces TIMP-1 mRNA Expression and ProteinRelease in Human Adherent Monocytes

Human adherent monocytes (16106/well) were left unfed

(control cells, CTR), fed with latex as a control meal (LATEX-

FED), or fed with nHZ (nHZ-FED) for 2 h. At the end of

phagocytosis cells were washed and incubated for 15 h. Thereaf-

ter, mRNA was extracted and TIMP-1 expression was measured

by Real Time RT-PCR (Figure 1A). nHZ enhanced .50-fold

TIMP-1 mRNA expression compared to CTR, while latex

particles did not significantly alter basal TIMP-1 mRNA levels.

Alternatively, to evaluate TIMP-1 protein secretion, monocytes

were washed and incubated for 24 h after phagocytosis, with cell

supernatants collected at selected time-points (0, 6, 15, and 24 h)

during the incubation. TIMP-1 protein levels were evaluated by

Western blotting and densitometry (24 h end-point studies,

Figure 1B) and by ELISA (0–24 h time course studies,

Figure 1C). TIMP-1 protein release was induced de novo in nHZ-

FED cells, whereas Western blotting analysis did not detect

apparent levels of TIMP-1 in 24 h supernatants of CTR and

LATEX-FED cells. To verify these results, we used a more

sensitive ELISA assay to examine levels of TIMP-1 at earlier time-

points. Over time, CTR cells released low levels of TIMP-1

protein (up to ,3,5 ng/ml at the end of the 24 h observational

period), which were significantly increased upon nHZ treatment in

the nHZ-FED cells at 15 h and 24 h post phagocytosis (up to

,10,5 ng/ml at the end of the 24 h observational period).

Notably, nHZ and latex did not display any cytotoxic effects on

monocytes, and did not affect cell viability (see Figure S1A–B).

We next questioned whether nHZ-FED monocytes could

modulate TIMP-1 secretion from unfed cells. To address this

possibility, we co-cultured both unfed and nHZ-fed monocytes in

six-well plate Transwell systems with 0.4 mm of porosity, plating

unfed monocytes (16106 cells/well) at the bottom of the wells and

seeding nHZ-fed human adherent monocytes (0,56106 cells/well)

onto the inserts. Co-cultures were incubated for 2 h before

removal of the inserts. After washing, unfed monocytes were

further incubated for 24 h. Then, cell supernatants were collected

and analysed for TIMP-1 secretion. Results (see Figure S2A) show

that unfed monocytes co-cultured with nHZ-fed monocytes secrete

higher amounts of TIMP-1 than control cells, albeit significantly

lower in comparison to the amounts released by nHZ-fed cells

cultured alone.

Role of TNF-a, IL-1b, and MIP-1a/CCL3 in nHZ-dependentTIMP-1 Induction

Protein levels of TNF-a, IL-1b, and MIP-1a/CCL3 released

from CTR, LATEX-FED and nHZ-FED monocytes (16106/well)

were measured over 24 h post phagocytosis by ELISA (Figure 2).

During the observational period, CTR and LATEX-FED cells

released low amounts of all three pro-inflammatory molecules in a

time-dependent manner, reaching less than 5 ng/ml TNF-a and

less than 3 ng/ml IL-1b or MIP-1a/CCL3 at 24 h as evaluated

for both conditions. No significant differences between CTR and

LATEX-FED cells were found. After phagocytosis of nHZ, the

production of pro-inflammatory molecules was significantly higher

than in CTR or LATEX-FED cells, reaching ,15 ng/ml TNF-aand ,12 ng/ml IL-1b and MIP-1a/CCL3 at 15 h, as well as

,22 ng/ml TNF-a or IL-1b and ,15 ng/ml MIP-1a/CCL3 at

24 h.

As a next step, a double blocking and mimicking approach was

used to investigate the role of IL-1b, TNF-a, and MIP-1a/CCL3

(alone or combined) in the nHZ-dependent induction of TIMP-

1 mRNA expression and protein release (Figure 3).

After the end of phagocytosis CTR and nHZ-FED cells

(16106/well) were left untreated or treated with single or

combined doses (30 ng/ml) of anti-hTNF-a, anti-hIL-1b and

anti-hMIP-1a/CCL3 blocking antibodies for 15–24 h. We ana-

lyzed TIMP-1 mRNA expression and protein release by Real

Time RT-PCR in cell lysates (15 h) and Western blotting in cell

supernatants (24 h), respectively. The effects of nHZ on either

mRNA expression or protein release were reduced by single

blocking antibodies, and were fully abrogated by the combination

of all three antibodies (Panels 3A and 3C). Neither basal TIMP-

1 mRNA nor TIMP-1 protein levels in CTR cells were affected by

any treatments with blocking antibodies and by non-immune Ig

used as a negative control (data not shown).

Alternatively, after the end of phagocytosis CTR and nHZ-FED

cells were left untreated or treated with single or combined doses

(20 ng/ml) of rhTNF-a, rhIL-1b and rhMIP-1a/CCL3. Again,

TIMP-1 mRNA expression and protein release were analyzed as

previously indicated. The effects of nHZ on either mRNA

expression or protein release were only partially mimicked by

adding the single recombinant molecules to CTR cells, whereas

they were fully recapitulated by the simultaneous addition of all

three recombinant molecules (Panels 3B and 3D). None of

treatments with recombinant molecules further induced TIMP-1

mRNA or protein levels in nHZ-FED cells, possibly because a

plateau was already reached after nHZ phagocytosis (data not

shown).

Of note, blocking antibodies and recombinant molecules did not

display any cytotoxic effects on monocytes and did not affect cell

viability (see Figure S1A-B).

Involvement of p38 MAPK Pathway in nHZ-inducedRelease of TIMP-1 and Pro-inflammatory Molecules

Human adherent monocytes (16106/well) were left unfed or fed

with nHZ for 2 h and then incubated for an additional 24 h in the

presence or absence of p38 MAPK inhibitor SB203580 (10 mM)

[34]. Supernatants were collected and TIMP-1 release was

evaluated by Western blotting and subsequent densitometry,

whereas TNF-a, IL-1b, and MIP-1a/CCL3 production was

measured by ELISA (Figure 4, panel A: TIMP-1 protein release;

panel B: production of pro-inflammatory molecules).

Addition of SB203580 abrogated nHZ-induced protein release

of TIMP-1, TNF-a, IL-1b, and MIP-1a/CCL3. SB203580

treatment did not affect basal protein levels of TIMP-1 or pro-

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inflammatory molecules in CTR cell supernatants (data not

shown). SB203580 did not display any cytotoxic effects on

monocytes and did not affect cell viability (see Figure S1A–B).

Involvement of NF-kB Pathway in nHZ-induced Releaseof TIMP-1 and Pro-inflammatory Molecules

Human adherent monocytes (16106/well) were left unfed or fed

with nHZ for 2 h and then incubated for additional 24 h in the

presence or absence of three inhibitory molecules which have been

reported to block NF-kB signaling at different levels: 15 mM

quercetin, inhibitor of I-kBa phosphorylation and subsequent

degradation [35]; 10 mM artemisinin, inhibitor of NF-kB nuclear

translocation [36]; and 10 mM parthenolide, inhibitor of NF-kB

binding to DNA [37]. After treatment with the denoted inhibitors,

cell supernatants were collected and TIMP-1 release was evaluated

by Western blotting and subsequent densitometry, while TNF-a,

IL-1b, and MIP-1a/CCL3 production was measured by ELISA

(Figure 5, panel A: TIMP-1 protein release; panel B: pro-

inflammatory molecules production).

nHZ-induced protein release of TIMP-1, TNF-a, IL-1b, and

MIP-1a/CCL3 was abrogated by all three NF-kB inhibitors.

Quercetin, artemisinin, and parthenolide did not affect basal

protein levels of TIMP-1 or pro-inflammatory molecules in CTR

cell supernatants (data not shown). NF-kB inhibitors did not

display any cytotoxic effects on monocytes, and did not affect cell

viability (see Figure S1A–B).

nHZ Enhances Total Gelatinolytic Activity of HumanMonocytes Despite TIMP-1 Induction

Unfed and nHZ-fed monocytes (16106/well) were incubated

for 24 h in the presence or absence of p38 MAPK or NF-kB

inhibitors. After collection of cell supernatants 6, 15, and 24 h

after phagocytosis, MMP-9 and TIMP-1 protein levels were

quantified by ELISA, and stoichiometric ratios were calculated.

Both MMP-9 and TIMP-1 levels were higher in nHZ-fed cell

supernatants compared to unfed cells at all time-points. However,

MMP-9/TIMP-1 stoichiometric ratios were .1 both in unfed and

nHZ-fed cell supernatants (with the exception of unfed cells at 6 h

of the observational period), suggesting that nHZ-dependent

TIMP-1 induction is not sufficient to counteract nHZ-dependent

MMP-9 increase (panels 6A–B).

We next sought to determine any overall net changes in

gelatinolytic activity by employing a fluorogenic gelatin conversion

assay [33] (Figure 6C). Consistently with data on MMP-9/TIMP-

1 stoichiometric ratios obtained by ELISA, total gelatinolytic

activity was enhanced after nHZ phagocytosis. As expected, p38

MAPK inhibitor and NF-kB inhibitors abrogated all nHZ effects

(panels 6B–C).

It is interesting to speculate if nHZ-fed monocytes can modulate

TIMP-1 and MMP-9 secretion of unfed cells. We tested this

possibility by co-culturing both unfed and nHZ-fed monocytes in

six-well plate Transwell systems and assessing changes in TIMP-1

and MMP-9 protein levels. Results show that unfed monocytes co-

cultured with nHZ-fed monocytes secrete higher amounts of

TIMP-1 and MMP-9 compared to controls, although the levels

are significantly lower compared to nHZ-fed cells alone (see Figure

S2B). Nevertheless, in all cases MMP-9/TIMP-1 ratios were .1,

confirming that nHZ-dependent TIMP-1 induction is not

sufficient to counteract nHZ-dependent MMP-9 increase.

Discussion

TIMP molecules are widely distributed in the animal kingdom,

with four paralogous genes encoding TIMP-1 to -4 in the human

genome [18]. TIMPs were originally characterized as endogenous

MMP inhibitors due to their ability to bind zinc in the MMP

active site and thereby antagonize the effects of activated/cleaved

MMPs and block enzyme activity. TIMPs are key regulators of

crucial MMP-related pathophysiological processes, such as turn-

over of extracellular matrix and shedding of cell surface molecules

[38]. Additionally, TIMPs have important roles in a broad

spectrum of biological activities often independent of MMPs,

including effects on cell survival, cell growth and differentiation,

cell migration, angiogenesis, and synaptic plasticity [39–40].

Figure 1. nHZ induces TIMP-1 mRNA expression and protein release in human adherent monocytes. Cells were left unfed (CTR), fed withlatex, or fed with nHZ for 2 h. Subsequently, monocytes were washed and incubated for 15 h, and TIMP-1 mRNA levels were measured in cell lysatesby Real Time RT-PCR (Panel A); alternatively, monocytes were washed and incubated for 24 h, and TIMP-1 protein levels were evaluated in 24 h cellsupernatants by Western blotting and densitometry (Panel B) or in 6, 15, and 24 h cell supernatants by ELISA (Panel C). Data are indicated as meanvalues+SEM or as a representative blot of three independent experiments. All data were evaluated for significance by ANOVA. Panel A: Vs CTR cells(column 1) *p,0.05; Vs LATEX-fed cells (column 2) up,0.05. Panel B: Vs CTR cells (column 1) *p,0.0001; Vs LATEX-fed cells (column 2) up,0.0001.Panel C: Vs CTR cells (black circled line) *p,0.0001.doi:10.1371/journal.pone.0071468.g001

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It has only recently come to light that TIMPs are involved in

malaria disease state. Currently, our understanding of TIMPs in

malaria manifests from only a few studies showing dysregulated

TIMP levels in different in vivo and in vitro malaria models. In vivo,

brain and spleen TIMP-1 mRNA levels are increased in CM-

sensitive mice infected with P. berghei ANKA [19]. Further,

increased TIMP-1 serum levels are associated with severe malaria

in Rhesus macaques infected with P. coatneyi [20]. Moreover, in a

case-control study on Gabonese children, TIMP-1 serum levels

were significantly higher in malaria patients than in healthy

controls and correlated with disease severity, suggesting a potential

role for TIMP-1 as a diagnostic marker [21]. In vitro, P. falciparum-

IRBCs and nHZ promoted TIMP-2 but not TIMP-1 protein

release from human microvascular endothelial cells [15–16]. On

the other hand, in human monocytes, nHZ did not affect TIMP-2

production [41], whereas no data on TIMP-1 regulation by nHZ

were available so far.

The present work explores the effects of phagocytosed nHZ on

monocyte TIMP-1 expression and release, and identifies cellular

mechanisms and soluble mediators involved. Untreated cells, used

as negative controls, express low TIMP-1 mRNA levels and

secrete negligible TIMP-1 protein, consistent with previous reports

[23,42]. Interestingly, 15 h after phagocytosis of nHZ, TIMP-

1 mRNA expression is significantly enhanced, and higher TIMP-1

protein levels are detected in cell supernatants up to 24 h post

nHZ phagocytosis. The effect is specific for nHZ and not due to

phagocytosis per se since latex particles do not induce TIMP-1

expression and secretion.

These data give insight into the molecular mechanisms that

enable nHZ to alter the phenotype of human monocytes. Indeed,

nHZ was previously shown to induce gene expression of a large

number of pro-inflammatory molecules including cytokines (IL-1b,

TNF-a, IL-1RA) and chemokines (MIP-1a/CCL3, MIP-1b/

CCL4, MCP-1/CCL2, IL-8/CXCL8, ENA-78/CXCL5,

GROa/CXCL1, GROb/CXCL2, GROc/CXCL3) [43]. Also,

nHZ was reported to promote expression and release of MMP-9

[11–13] and lysozyme [24,31]. Notably, it has been proposed that

IL-1b, TNF-a and MIP-1a/CCL3 function as soluble mediators

for nHZ-dependent upregulation of either MMP-9 [11–12,14] or

lysozyme [24,31]. Here we show that these three pro-inflamma-

tory molecules are also involved in nHZ-induced expression and

release of TIMP-1. Either 15 or 24 h after phagocytosis of nHZ,

the levels of TNF-a, IL-1b, and MIP-1a/CCL3 in cell superna-

tants are significantly higher compared to controls. Treating with

blocking antibodies directed against these three molecules

abrogates the effects of nHZ on monocyte TIMP-1 mRNA

expression and protein release. Consistently, unfed cells treated

with recombinant TNF-a, IL-1b, and MIP-1a/CCL3 display

TIMP-1 mRNA levels in lysates and TIMP-1 protein levels in

supernatants similar to those of nHZ-fed cells. These results are in

line with previous data showing TIMP-1 induction by IL-1b [44].

Apparently, all three molecules are required in combination to

obtain nHZ-dependent TIMP-1 induction, since only partial

abrogation/emulation of nHZ effects is obtained with single doses

of blocking antibodies/recombinant molecules, whereas the effect

is fully reached after using combined doses. Nevertheless, nHZ

induces a plethora of pro-inflammatory molecules [43], some of

which correlate with TIMP-1 gene expression (i.e. MCP-1/CCL2,

MIP-1b/CCL4, and RANTES/CCL5) [45–46]. Therefore, the

involvement of additional cytokines and chemokines as redundant

soluble mediators for nHZ-promoted TIMP-1 expression and

release cannot be excluded.

We next wanted to investigate the molecular mechanisms

underlying nHZ-induced upregulation of TIMP-1 and related pro-

inflammatory molecules. It has been previously reported that IL-

1b induces both TIMP-1 and MMP-9 in monocytes and

monocytic leukemia cells [44,47]. In a separate study using

human monocytes, gene expression of TIMP-1, MMP-9 and

TNF-a, required p38 MAPK and NF-kB pathway activation [48–

49]. Interestingly, several in vitro and in vivo studies have raised the

possibility that either MAPK or NF-kB routes are involved in

malaria.

To date we understand very little about nHZ-dependent

activation of MAPKs. However, recent emerging evidence in a

murine malaria model shows that nHZ induces the activation of

p38 [49] and ERK1/2 [50–53] MAPKs, but does not induce the

activation of other signaling pathways such as the JNK-2/STAT

pathway [47–49]. Evidence from a human malaria model shows

Figure 2. nHZ enhances production of TNF-a, IL-1b, and MIP-1a/CCL3 by human adherent monocytes. Cells were left unfed(CTR), fed with latex particles (LATEX), or fed with nHZ (nHZ) for 2 h;therefore, production of TNF-a (upper panel), IL-1b (central panel), andMIP-1a/CCL3 (lower panel) was monitored in cell supernatants 0, 15 and24 h after the end of phagocytosis. Data are means+SEM of threeindependent experiments. Production of pro-inflammatory molecules isindicated as ng/ml. All data were evaluated for significance by ANOVA.Vs control cells (CTR) *p,0.0001; Vs LATEX-fed cells (LATEX) up,0.0001.doi:10.1371/journal.pone.0071468.g002

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nHZ activates p38 MAPK signaling, similar to the murine model,

whilst nHZ does not appear to activate the ERK1/2 or JNK-1/2

signaling pathways in the human model [25,31,54]. Interestingly,

a study using syncytiotrophoblast cells shows nHZ-dependent

phosphorylation of ERK1/2, along with IRBC-dependent phos-

phorylation of JNK-1, with both events being causally related to

production of pro-inflammatory molecules (TNF-a, MIP-1a/

CCL3, IL-8/CXCL8) [55–56]. Moreover, our group recently

showed intravenous injection of nHZ in malaria-free mice induces

an inflammatory frame similar to that observed in experimental

malaria-associated acute respiratory distress syndrome, pointing to

nHZ as a prominent inflammatory virulent factor in lung

pathology [26]. On the other hand, Plasmodium falciparum

glycosylphosphatidylinositol (PfGPI) promotes phosphorylation of

all major MAPK routes, including p38, ERK1/2, and JNK-2/

STAT-1 [57–58]. Inhibition of PfGPI-dependent activation of

MAPKs decreases inflammatory responses and enhances phago-

cytic clearance of IRBCs in mice infected by Plasmodium berghei or

chabaudi chabaudi [59]. Future research defining the differential

roles of nHZ, IRBCs, and GPI in regulating MAPKs and

inflammation in malaria, both in vitro and in vivo, will be certainly

welcomed.

Along with nHZ-dependent regulation of MAPKs, emerging

evidence suggests malarial pigment plays a role in activating the

NF-kB pathway. In murine macrophages fed with nHZ or sHZ,

NF-kB activation is required to upregulate expression of inducible

nitric oxide synthase [51] and production of several chemokines,

including MIP-1a/CCL3, MIP-1b/CCL4, MIP-2/CXCL2, and

MCP-1/CCL2 [52]. Gene expression profiling of sHZ-laden

RAW 264.7 macrophage cells display altered NF-kB signal

transduction, enhanced inflammatory response and a severe

MMP-9/TIMP-1 imbalance in favor of ECM proteolysis [60].

NF-kB activation is also mandatory for nHZ-induced MMP-9

expression in human THP-1 monocyte cell line [61] and for nHZ-

Figure 3. Role of TNF-a, IL-1b, and MIP-1a/CCL3 in nHZ-induced mRNA expression and protein release of human monocytic TIMP-1.Human adherent monocytes were left unfed (negative controls) or fed with nHZ (positive controls). Thereafter, cells were incubated for 15 h (mRNAstudies, Panels A-B) or 24 h (protein studies, Panels C-D) in the presence/absence of the following treatments: single or combined doses (30 ng/ml) ofanti-hTNF-a, anti-hIL-1b and anti-hMIP-1a/CCL3 blocking antibodies (Panels A and C, blocking approach); and single or combined doses (20 ng/ml) ofrhTNF-a, rhIL-1b and rhMIP-1a/CCL3 (Panels B and D, mimicking approach). After lysis, TIMP-1 mRNA expression was measured by Real Time RT-PCR;alternatively, TIMP-1 protein levels in cell supernatants were evaluated by Western blotting and densitometry. Data are indicated as meanvalues+SEM or as a representative blot of three independent experiments. All data were evaluated for significance by ANOVA. Panel A: Vsunstimulated cells (column 1) *p,0.01; Vs untreated nHZ-fed cells (column 2) up,0.05. Panel B: Vs unstimulated cells (column 1) *p,0.05, **p,0.01.Panel C: Vs unstimulated cells (column 1) *p,0.05, **p,0.0001; Vs untreated nHZ-fed cells (column 2) up,0.01, uup,0.0001; Vs anti-TNFa-treatednHZ-fed cells (column 3) 1p,0.05. Panel D: Vs unstimulated cells (column 1) *p,0.05, **p,0.01, ***p,0.001.doi:10.1371/journal.pone.0071468.g003

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Figure 4. Involvement of p38 MAPK pathway in nHZ-induced release of TIMP-1 and related pro-inflammatory molecules fromhuman adherent monocytes. Cells were left unfed (negative controls) or fed with nHZ (positive controls) for 2 h; after phagocytosis, cells wereincubated for 24 h alone or with 10 mM SB203580 (p38 MAPK synthetic inhibitor). Thereafter, cell supernatants were collected. TIMP-1 protein releasewas analyzed by Western blotting and densitometry (Panel A); TNF-a, IL-1b, and MIP-1a/CCL3 production was measured by ELISA (Panel B). Data areindicated as mean values+SEM or as a representative blot of three independent experiments. All data were evaluated for significance by ANOVA.Panel A: Vs unstimulated cells (column 1) *p,0.0001; Vs untreated nHZ-fed cells (column 2) up,0.0001. Panel B: Vs unstimulated cells (dataset 1)*p,0.0001; Vs untreated nHZ-fed cells (dataset 2) up,0.01, uup,0.0001.doi:10.1371/journal.pone.0071468.g004

Figure 5. Involvement of NF-kB pathway in nHZ-induced release of TIMP-1 and related pro-inflammatory molecules from humanadherent monocytes. Cells were left unfed (negative controls) or fed with nHZ (positive controls) for 2 h. After phagocytosis, cells were incubatedfor 24 h alone or with 15 mM quercetin, 10 mM artemisinin and 10 mM parthenolide (NF-kB inhibitors). Thereafter, cell supernatants were collected.TIMP-1 protein release was analyzed by Western blotting and densitometry (Panel A); TNF-a, IL-1b, and MIP-1a/CCL3 production was measured byELISA (Panel B). Data are indicated as mean values+SEM or as a representative blot of three independent experiments. All data were evaluated forsignificance by ANOVA. Panel A: Vs unstimulated cells (column 1) *p,0.0001; Vs untreated nHZ-fed cells (column 2) u p,0.0001. Panel B: Vsunstimulated cells (dataset 1) *p,0.0001; Vs untreated nHZ-fed cells (dataset 2) u p,0.0001.doi:10.1371/journal.pone.0071468.g005

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enhanced release of MMP-9, lysozyme, TNF-a and IL-1b from

human adherent monocytes isolated from peripheral blood

[13,25,31]. Consistently, in peripheral mononuclear cells of

malaria patients, phospho-NF-kB p65 levels are significantly

higher than in healthy controls [62], and genome wide expression

profiles display NF-kB-dependent enhancement of inflammatory

cytokines [63].

Results from the present work suggest that nHZ-induced

secretion of TIMP-1 and related pro-inflammatory molecules

from human monocytes require the involvement of either p38

MAPK- or NF-kB-dependent mechanisms. Indeed, the effects of

nHZ-mediated protein release of TIMP-1, TNF-a, IL-1b and

MIP-1a/CCL3 are fully abrogated by using micromolar doses of

SB203580, a synthetic inhibitor of p38 MAPK pathway [34], or

after treatment with quercetin, artemisinin and parthenolide -

three molecules showing antimalarial properties [64–65] able to

block NF-kB pathway at different levels (I-kBa phosphorylation

and subsequent degradation [35]; NF-kB nuclear translocation

[36]; and NF-kB binding to DNA [37], respectively). Interestingly,

these molecules also abrogate lysozyme and MMP-9 release in the

same cellular model [13,25,31,54].

Collectively, the present data show phagocytosis of nHZ by

human monocytes induces inflammation-mediated expression and

release of TIMP-1 through p38 MAPK- and NF-kB-dependent

mechanisms, suggesting that in vivo nHZ-fed human monocytes

may be a source for the high TIMP-1 serum levels found by

Dietmann and colleagues in malaria patients [21]. In this context,

TIMP-1 levels are also related to disease severity. However, it

remains unclear how TIMP-1 may exacerbate the clinical course

in malaria patients. One major function of TIMP-1 is to

counteract and modulate the numerous effects of MMP-9, which

include degradation of the sub-endothelial basal lamina, modula-

tion of the activity of several pro-inflammatory molecules,

disruption of tight junctions, and impairment of haemostasis [4–

7;38], all of which comprise key roles in CM. In this way it is

attractive to hypothesize that increased TIMP-1 levels are actually

protective rather than a risk factor in malaria prognosis, as they

could contrast the potentially detrimental effects of nHZ-enhanced

MMP-9. However, our results show that MMP-9/TIMP-1

stoichiometric ratios and total gelatinolytic activity measured in

nHZ-fed monocyte supernatants were significantly higher than in

controls, arguing against TIMP-1 ability to act as a protective

factor in this context. Total gelatinolytic activity reflects the net

overall activity of gelatinases (MMP-2 and -9) and their

endogenous inhibitors (TIMP-2 and TIMP-1, respectively) [33],

but in the present model it should be noted that the activity is due

solely to the net balance between MMP-9 and TIMP-1, as unfed

and nHZ-fed human adherent monocytes do not release MMP-2

and TIMP-2 proteins [41]. Therefore, these results suggest that

nHZ-dependent induction of TIMP-1 expression and release is not

sufficient to counterbalance nHZ-enhanced MMP-9 levels re-

leased from human monocytes.

Intriguingly, TIMP-1 could play a detrimental role through an

MMP-independent mechanism [39–40]. MMP-independent roles

Figure 6. nHZ-dependent induction of TIMP-1 secretion does not counterbalance nHZ-dependent MMP-9 enhancement in humanadherent monocytes. Cells were left unfed or fed with nHZ for 2 h; after washing, cells were incubated for 24 h alone or with 10 mM SB203580(p38 MAPK synthetic inhibitor), 15 mM quercetin, 10 mM artemisinin and 10 mM parthenolide (NF-kB inhibitors). During the incubation, cellsupernatants were collected at different time-points (6, 15 and 24 h). Thereafter, MMP-9 and TIMP-1 protein levels were measured by ELISA, andMMP-9/TIMP-1 ratios were calculated; additionally, total gelatinolytic activity was analyzed by fluorogenic gelatin conversion assay. Panel A. MMP-9/TIMP-1 stoichiometric ratios in unfed (white columns) and nHZ-fed (black columns) cell supernatants during time (6, 15 and 24 after the end ofphagocytosis). Panel B. MMP-9 (white columns) and TIMP-1 (black columns) protein levels (expressed as pmol/ml) in unfed and nHZ-fed cellsupernatants, in the presence/absence of p38 MAPK and NF-kB inhibitors. Panel C. Gelatinolytic activity (expressed as gelatinolytic activity units perml, with one gelatinolytic unit corresponding to 37 ng activated rhMMP-9) in unfed and nHZ-fed cell supernatants, in the presence/absence of p38MAPK and NF-kB inhibitors. Data are mean values+SEM of three independent experiments. All data were evaluated for significance by ANOVA. PanelA: Vs unfed cells (6 h) *p,0.05, **p,0.005; Vs nHZ-fed cells (6 h) up,0.001, uup,0.0001; Vs unfed cells (15 h) #p,0.0001. Panel B: Vs unstimulatedcells (column 1) *p,0.0001; Vs untreated nHZ-fed cells (column 2) up,0.0001. Panel C: Vs unstimulated cells (column 1) *p,0.05; Vs untreated nHZ-fed cells (column 2) up,0.05.doi:10.1371/journal.pone.0071468.g006

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for TIMP-1 in biological processes include anti-apoptotic effects of

TIMP-1 in several human cells, such as Burkitt’s lymphoma [66],

breast epithelial cells [67], and cardiomyocytes [68]. Recently

CD63, a member of the tetraspanin family, was identified as a cell-

binding partner for TIMP-1 in human mammary epithelial cells,

and CD63 down-regulation with shRNA resulted in reduced

TIMP-1 binding and restored cell apoptosis [69]. Interestingly,

nHZ-fed monocytes do not undergo apoptosis, despite increased

inflammation and functional impairment [30,43]. CD63 is

constitutively expressed by human monocytes [70]. Thus, it is

intriguing to speculate that nHZ-enhanced TIMP-1 levels might

prevent apoptosis of functionally impaired nHZ-fed cells through

CD63-dependent mechanisms. Additionally, there are several

reports describing MMP-independent abilities of TIMP-1 to

inhibit cell growth and differentiation [39–40,71]. These TIMP-

1 properties may be crucial for nHZ-fed monocytes, since these

cells have been shown not to mature to dendritic cells [72] and not

to coordinate erythropoiesis [73].

In conclusion, the present work expands the available evidence

on the expression of TIMPs in malaria, providing new information

on the mechanisms underlying nHZ-dependent TIMP-1 increase.

Future investigation is needed to ascertain whether nHZ-enhanced

TIMP-1 may contribute to worsen the clinical course in malaria

patients as a consequence of its MMP-independent anti-apoptotic

or growth/differentiation-inhibitory properties. Further, previous

evidence correlating the number of circulating nHZ-laden

monocytes in patients to parasitaemia degree and malaria severity

[29] seems to support such a hypothesis. More extensive research

on the functional role of TIMP-1 in malaria, along with a better

understanding of MMP-independent TIMP functions is necessary

in order to find new tools for differential diagnosis and therapy of

severe malaria.

Supporting Information

Figure S1 Phagocytic meals and treatments do notdisplay cytotoxicity and do not affect viability of humanadherent monocytes. Cells were left unfed or fed with nHZ

and latex for 2 h; after washing, nHZ-fed cells were incubated for

24 h alone, whereas unfed cells were incubated for 24 h with

30 ng/ml of anti-hTNF-a, anti-hIL-1b, anti-hMIP-1a/CCL3

blocking antibodies; 20 ng/ml of rhTNF-a, rhIL-1b, rhMIP-1a/

CCL3; 10 mM SB203580; 15 mM quercetin; 10 mM artemisinin;

and 10 mM parthenolide. Thereafter, cell supernatants and lysates

were collected and LDH activity was measured by a spectrometric

assay. Panel A. Cytotoxicity of phagocytic meals and treatments,

expressed as percentage of (extracellular LDH activity)/(total

LDH activity) ratio versus controls (unfed/untreated monocytes).

Panel B. Viability of cells after exposure to phagocytic meals and

treatments, expressed as percentage of (intracellular LDH

activity)/(total LDH activity) ratio versus controls (unfed untreated

monocytes). Data are mean values+SEM of three independent

experiments. All data were evaluated for significance by ANOVA:

no significant differences were found.

(TIF)

Figure S2 Co-culturing with nHZ-fed monocytes induc-es unfed cells to release TIMP-1 and MMP-9. Unfed

monocytes (16106 cells/well) were plated at the bottom of the

wells, with nHZ-fed human adherent monocytes (0,56106 cells/

well) seeded onto the inserts. Co-cultures were incubated for 2 h

before removal of the inserts. After washings, unfed monocytes

were further incubated for 24 h. Non-co-cultured unfed and nHZ-

cells were also used as negative and positive controls, respectively.

Cell supernatants were collected and analysed for TIMP-1 and

MMP-9 secretion by ELISA. Panel A. Secretion of TIMP-1 (white

columns), expressed as pg/ml. Panel B. Secretion of MMP-9

(white columns) and TIMP-1 (black columns), expressed as pmol/

ml. Data are mean values+SEM of three independent experi-

ments. All data were evaluated for significance by ANOVA. Panel

A: Vs non-co-cultured unfed cells (column 1) *p,0.0001; Vs non-

co-cultured nHZ-fed cells (column 3) up,0.0001. Panel B: Vs non-

co-cultured unfed cells (column 1) *p,0.0001.

(TIF)

Acknowledgments

Thanks are due to Nathalie Geurts and Erik Martens for help with the

fluorogenic gelatin conversion assay; to Valentina Gallo for help with

FACS and Real Time RT-PCR analyses; to Giulia Rossana Gulino for

help with LDH experiments; to Nicoletta Basilico and colleagues for

providing supplementary nHZ and six-well plate Transwell systems for co-

culturing experiments; to Mary Lynn Dear and Andrew Giles for

comments on the manuscript; and to Associazione Volontari Italiani

Sangue (AVIS Torino) for providing fresh blood.

Author Contributions

Conceived and designed the experiments: GG M. Prato. Performed the

experiments: M. Polimeni EV DU GG M. Prato. Analyzed the data: M.

Polimeni GO PEVDS GG M. Prato. Contributed reagents/materials/

analysis tools: EV GO PEVDS GG M. Prato. Wrote the paper: M.

Polimeni GG M. Prato.

References

1. Khadjavi A, Giribaldi G, Prato M (2010) From control to eradication of malaria:

the end of being stuck in second gear? Asian Pac J Trop Med 3: 412–420.

2. WHO (2011) World Malaria Report.

3. Higgins SJ, Kain KC, Liles WC (2011) Immunopathogenesis of falciparum

malaria: implications for adjunctive therapy in the management of severe and

cerebral malaria. Expert Rev Anti Infect Ther 9: 803–819.

4. Lucchi NW, Jain V, Wilson NO, Singh N, Udhayakumar V, et al (2011)

Potential serological biomarkers of cerebral malaria. Dis Markers 31: 327–335.

5. Nagase H, Woessner JF (1999) Matrix metalloproteinases. J Biol Chem 274:

21491–21494.

6. Cauwe B, Van den Steen PE, Opdenakker G (2007) The biochemical,

biological, and pathological kaleidoscope of cell surface substrates processed by

matrix metalloproteinases. Crit Rev Biochem Mol Biol 42: 113–185.

7. Seo JH, Guo S, Lok J, Navaratna D, Whalen MJ, et al (2012) Neurovascular

matrix metalloproteinases and the blood-brain barrier. Curr Pharm Des 18:

3645–3648.

8. Szklarczyk A, Stins M, Milward EA, Ryu H, Fitzsimmons C, et al (2007) Glial

activation and matrix metalloproteinase release in cerebral malaria. J Neurovirol

13: 2–10.

9. Prato M, Giribaldi G (2011b) Matrix Metalloproteinase-9 and Haemozoin:

Wedding Rings for Human Host and Plasmodium falciparum Parasite inComplicated Malaria. J Trop Med 2011: 628435.

10. Geurts N, Opdenakker G, Van den Steen PE (2012) Matrix metalloproteinases

as therapeutic targets in protozoan parasitic infections. Pharmacol Ther 133:

257–279.

11. Prato M, Giribaldi G, Polimeni M, Gallo V, Arese P (2005) Phagocytosis ofhemozoin enhances matrix metalloproteinase-9 activity and TNF-alpha

production in human monocytes: role of matrix metalloproteinases in thepathogenesis of falciparum malaria. J Immunol 175: 6436–6442.

12. Prato M, Gallo V, Giribaldi G, Arese P (2008) Phagocytosis of haemozoin(malarial pigment) enhances metalloproteinase-9 activity in human adherent

monocytes: role of IL-1beta and 15-HETE. Malar J 7: 157.

13. Prato M, Gallo V, Giribaldi G, Aldieri E, Arese P (2010a) Role of the NF-kBtranscription pathway in the haemozoin- and 15-HETE-mediated activation of

matrix metalloproteinase-9 in human adherent monocytes. Cell Microbiol 12:

1780–1791.

14. Giribaldi G, Valente E, Khadjavi A, Polimeni M, Prato M (2011) Macrophageinflammatory protein-1alpha mediates matrix metalloproteinase-9 enhancement

in human adherent monocytes fed with malarial pigment. Asian Pac J Trop Med

4: 925–930.

Haemozoin Induces Human Monocyte TIMP-1

PLOS ONE | www.plosone.org 10 August 2013 | Volume 8 | Issue 8 | e71468

15. Prato M, D’Alessandro S, Van den Steen PE, Opdenakker G, Arese P, et al

(2011a) Natural haemozoin modulates matrix metalloproteinases and inducesmorphological changes in human microvascular endothelium. Cell Microbiol

13: 1275–1285.

16. D’Alessandro S, Basilico N, Prato M (2013) Effects of Plasmodium falciparum-infected erythrocytes on matrix metalloproteinase-9 regulation in human

microvascular endothelial cells. Asian Pac J Trop Med 6: 195–199.

17. Geurts N, Martens E, Van Aelst I, Proost P, Opdenakker G, et al (2008) Beta-

hematin interaction with the hemopexin domain of gelatinase B/MMP-9provokes autocatalytic processing of the propeptide, thereby priming activation

by MMP-3. Biochemistry 47: 2689–2699.

18. Brew K, Nagase H (2010) The tissue inhibitors of metalloproteinases (TIMPs):an ancient family with structural and functional diversity. Biochim Biophys Acta

1803: 55–71.

19. Van den Steen PE, Van Aelst I, Starckx S, Maskos K, Opdenakker G, et al

(2006b) Matrix metalloproteinases, tissue inhibitors of MMPs and TACE inexperimental cerebral malaria. Lab Invest 86: 873–888.

20. Moreno A, Cabrera-Mora M, Garcia A, Orkin J, Strobert E, et al (2013)

Plasmodium coatneyi in Rhesus Macaques Replicates the MultisystemicDysfunction of Severe Malaria in Humans. Infect Immun 81: 1889–1904.

21. Dietmann A, Helbok R, Lackner P, Issifou S, Lell B, et al (2008) Matrix

metalloproteinases and their tissue inhibitors (TIMPs) in Plasmodium falciparummalaria: serum levels of TIMP-1 are associated with disease severity. J Infect Dis

197: 1614–1620.

22. Hanemaaijer R, Koolwijk P, le Clercq L, de Vree WJ, van Hinsbergh VW

(1993) Regulation of matrix metalloproteinase expression in human vein andmicrovascular endothelial cells. Effects of tumour necrosis factor alpha,

interleukin 1 and phorbol ester. Biochem J 296 (Pt 3): 803–809.

23. Zhang Y, McCluskey K, Fujii K, Wahl LM (1998) Differential regulation ofmonocyte matrix metalloproteinase and TIMP-1 production by TNF-alpha,

granulocyte-macrophage CSF, and IL-1 beta through prostaglandin-dependent

and -independent mechanisms. J Immunol 161: 3071–3076.

24. Van den Steen PE, Van Aelst I, Hvidberg V, Piccard H, Fiten P, et al (2006a)The hemopexin and O-glycosylated domains tune gelatinase B/MMP-9

bioavailability via inhibition and binding to cargo receptors. J Biol Chem 281:18626–18637.

25. Polimeni M, Valente E, Aldieri E, Khadjavi A, Giribaldi G, et al (2012)

Haemozoin induces early cytokine-mediated lysozyme release from human

monocytes through p38 MAPK- and NF-kappaB-dependent mechanisms. PLoSOne 7: e39497.

26. Deroost K, Tyberghein A, Lays N, Noppen S, Schwarzer E, et al (2013)

Hemozoin induces lung inflammation and correlates with malaria-associatedacute respiratory distress syndrome. Am J Resp Cell Mol Biol 48: 589–600.

27. Schwarzer E, Turrini F, Ulliers D, Giribaldi G, Ginsburg H, et al (1992)

Impairment of macrophage functions after ingestion of Plasmodium falciparum-

infected erythrocytes or isolated malarial pigment. J Exp Med 176: 1033–1041.

28. Schwarzer E, Kuhn H, Valente E, Arese P (2003) Malaria-parasitizederythrocytes and hemozoin nonenzymatically generate large amounts of

hydroxy fatty acids that inhibit monocyte functions. Blood 101: 722–728.

29. Were T, Davenport GC, Yamo EO, Hittner JB, Awandare GA, et al (2009)Naturally acquired hemozoin by monocytes promotes suppression of RANTES

in children with malarial anemia through an IL-10-dependent mechanism.

Microbes Infect 11: 811–819.

30. Prato M, Gallo V, Valente E, Khadjavi A, Mandili G, et al (2010b) Malarialpigment enhances Heat Shock Protein-27 in THP-1 cells: new perspectives for

in vitro studies on monocyte apoptosis prevention. Asian Pac J Trop Med 3:934–938.

31. Polimeni M, Valente E, Aldieri E, Khadjavi A, Giribaldi G, et al (2013) Role of

15-HETE in haemozoin-induced lysozyme release from human adherent

monocytes. Biofactors 39: 304–314.

32. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29: e45.

33. Vandooren J, Geurts N, Martens E, Van den Steen PE, Jonghe SD, et al (2011)

Gelatin degradation assay reveals MMP-9 inhibitors and function of O-glycosylated domain. World J Biol Chem 2: 14–24.

34. Lee JC, Kumar S, Griswold DE, Underwood DC, Votta BJ, et al (2000)

Inhibition of p38 MAP kinase as a therapeutic strategy. Immunopharmacol 47:

185–201.

35. Nair MP, Mahajan S, Reynolds JL, Aalinkeel R, Nair H, et al (2006) Theflavonoid quercetin inhibits proinflammatory cytokine (tumor necrosis factor

alpha) gene expression in normal peripheral blood mononuclear cells viamodulation of the NF-kappa beta system. Clin Vaccine Immunol 13: 319–328.

36. Aldieri E, Atragene D, Bergandi L, Riganti C, Costamagna C, et al (2003)

Artemisinin inhibits inducible nitric oxide synthase and nuclear factor NF-kBactivation. FEBS Lett 552: 141–144.

37. Garcıa-Pineres AJ, Castro V, Mora G, Schmidt TJ, Strunck E, et al (2001)Cysteine 38 in p65/NF-kappaB plays a crucial role in DNA binding inhibition

by sesquiterpene lactones. J Biol Chem 276: 39713–39720.

38. Nagase H, Visse R, Murphy G (2006) Structure and function of matrixmetalloproteinases and TIMPs. Cardiovasc Res 69: 562–573.

39. Chirco R, Liu XW, Jung KK, Kim HR (2006) Novel functions of TIMPs in cell

signaling. Cancer Metastasis Rev 25: 99–113.

40. Stetler-Stevenson WG (2008) Tissue inhibitors of metalloproteinases in cell

signaling: metalloproteinase-independent biological activities. Sci Signal 1: re6.

41. Prato M (2011) Malarial pigment does not induce MMP-2 and TIMP-2 protein

release by human monocytes. Asian Pac J Trop Med 4: 756.

42. Bar-Or A, Nuttall RK, Duddy M, Alter A, Kim HJ, et al (2003) Analyses of all

matrix metalloproteinase members in leukocytes emphasize monocytes as major

inflammatory mediators in multiple sclerosis. Brain 126: 2738–2749.

43. Giribaldi G, Prato M, Ulliers D, Gallo V, Schwarzer E, et al (2010) Involvement

of inflammatory chemokines in survival of human monocytes fed with malarial

pigment. Infect Immun 78: 4912–4921.

44. Opdenakker G, Masure S, Proost P, Billiau A, van Damme J (1991b) Natural

human monocyte gelatinase and its inhibitor. FEBS Lett 284: 73–78.

45. Yamamoto T, Eckes B, Mauch C, Hartmann K, Krieg T (2000) Monocyte

chemoattractant protein-1 enhances gene expression and synthesis of matrix

metalloproteinase-1 in human fibroblasts by an autocrine IL-1 alpha loop.

J Immunol 164: 6174–6179.

46. Johnatty RN, Taub DD, Reeder SP, Turcovski-Corrales SM, Cottam DW, et al

(1997) Cytokine and chemokine regulation of proMMP-9 and TIMP-1

production by human peripheral blood lymphocytes. J Immunol 158: 2327–

2333.

47. Opdenakker G, Masure S, Grillet B, Van Damme J (1991a) Cytokine-mediated

regulation of human leukocyte gelatinases and role in arthritis. Lymphokine

Cytokine Res 10: 317–324.

48. Nguyen J, Gogusev J, Knapnougel P, Bauvois B (2006) Protein tyrosine kinase

and p38 MAP kinase pathways are involved in stimulation of matrix

metalloproteinase-9 by TNF-alpha in human monocytes. Immunol Lett 106:

34–41.

49. Jovanovic DV, Di Battista JA, Martel-Pelletier J, Reboul P, He Y, et al (2001)

Modulation of TIMP-1 synthesis by antiinflammatory cytokines and prosta-

glandin E2 in interleukin 17 stimulated human monocytes/macrophages.

J Rheumatol 28: 712–718.

50. Cambos M, Bazinet S, Abed E, Sanchez-Dardon J, Bernard C, et al (2010) The

IL-12p70/IL-10 interplay is differentially regulated by free heme and hemozoin

in murine bone-marrow-derived macrophages. Int J Parasitol 40: 1003–1012.

51. Jaramillo M, Gowda DC, Radzioch D, Olivier M (2003) Hemozoin increases

IFN-gamma-inducible macrophage nitric oxide generation through extracellular

signal-regulated kinase- and NF-kappa B-dependent pathways. J Immunol 171:

4243–4253.

52. Jaramillo M, Godbout M, Olivier M (2005) Hemozoin induces macrophage

chemokine expression through oxidative stress-dependent and -independent

mechanisms. J Immunol 2005. 174: 475–484.

53. Griffith JW, Sun T, McIntosh MT, Bucala R (2009) Pure Hemozoin is

inflammatory in vivo and activates the NALP3 inflammasome via release of uric

acid. J Immunol 183: 5208–5220.

54. Khadjavi A, Valente E, Giribaldi G, Prato M (2013) Involvement of p38 MAPK

in natural haemozoin- and 15-HETE-dependent MMP-9 enhancement in

human adherent monocytes. Cell Biochem Funct. Epub 2013 Mar 7. DOI:

10.1002/cbf.2963.

55. Lucchi NW, Peterson DS, Moore JM (2008) Immunologic activation of human

syncytiotrophoblast by Plasmodium falciparum. Malar J 7: 42.

56. Lucchi NW, Sarr D, Owino SO, Mwalimu SM, Peterson DS, et al (2011)

Natural hemozoin stimulates syncytiotrophoblast to secrete chemokines and

recruit peripheral blood mononuclear cells. Placenta 32: 579–585.

57. Zhu J, Wu X, Goel S, Gowda NM, Kumar S, et al (2009) MAPK-activated

protein kinase 2 differentially regulates plasmodium falciparum glycosylpho-

sphatidylinositol-induced production of tumor necrosis factor-{alpha} and

interleukin-12 in macrophages. J Biol Chem 284: 15750–15761.

58. Lu Z, Serghides L, Patel SN, Degousee N, Rubin BB, et al (2006) Disruption of

JNK2 decreases the cytokine response to Plasmodium falciparum glycosylpho-

sphatidylinositol in vitro and confers protection in a cerebral malaria model.

J Immunol 177: 6344–6352.

59. Serghides L, Patel SN, Ayi K, Lu Z, Gowda DC, et al (2009) Rosiglitazone

modulates the innate immune response to Plasmodium falciparum infection and

improves outcome in experimental cerebral malaria. J Infect Dis 199: 1536–

1545.

60. Schrimpe AC, Wright DW (2009) Comparative analysis of gene expression

changes mediated by individual constituents of hemozoin. Chem Res Toxicol

22: 433–445.

61. Dell’agli M, Galli GV, Bulgari M, Basilico N, Romeo S, et al (2010)

Ellagitannins of the fruit rind of pomegranate (Punica granatum) antagonize

in vitro the host inflammatory response mechanisms involved in the onset of

malaria. Malar J 9: 208. DOI: 10.1186/1475-2875-9-208.

62. Punsawad C, Krudsood S, Maneerat Y, Chaisri U, Tangpukdee N, et al (2012)

Activation of nuclear factor kappa B in peripheral blood mononuclear cells from

malaria patients. Malar J 11: 191.

63. Ockenhouse CF, Hu WC, Kester KE, Cummings JF, Stewart A, et al (2006)

Common and divergent immune response signaling pathways discovered in

peripheral blood mononuclear cell gene expression patterns in presymptomatic

and clinically apparent malaria. Infect Immun 74: 5561–5573.

64. Burrows JN, Chibale K, Wells TN (2011) The state of the art in anti-malarial

drug discovery and development. Curr Top Med Chem 11: 1226–1254.

65. Khalid SA, Farouk A, Geary TG, Jensen JB (1986) Potential antimalarial

candidates from African plants: and in vitro approach using Plasmodium

falciparum. J Ethnopharmacol 15: 201–209.

Haemozoin Induces Human Monocyte TIMP-1

PLOS ONE | www.plosone.org 11 August 2013 | Volume 8 | Issue 8 | e71468

66. Guedez L, Stetler-Stevenson WG, Wolff L, Wang J, Fukushima P, et al (1998) In

vitro suppression of programmed cell death of B cells by tissue inhibitor of

metalloproteinases-1. J Clin Invest 102: 2002–2010.

67. Li G, Fridman R, Kim HR (1999) Tissue inhibitor of metalloproteinase-1

inhibits apoptosis of human breast epithelial cells. Cancer Res 59: 6267–6275.

68. Singla DK, McDonald DE (2007) Factors released from embryonic stem cells

inhibit apoptosis of H9c2 cells. Am J Physiol Heart Circ Physiol 293: H1590–

1595.

69. Jung KK, Liu XW, Chirco R, Fridman R, Kim HR (2006) Identification of

CD63 as a tissue inhibitor of metalloproteinase-1 interacting cell surface protein.

EMBO J 25: 3934–3942.

70. Pols MS, Klumperman J (2009) Trafficking and function of the tetraspanin

CD63. Exp Cell Res 315: 1584–1592.71. Egea V, Zahler S, Rieth N, Neth P, Popp T, et al (2012) Tissue inhibitor of

metalloproteinase-1 (TIMP-1) regulates mesenchymal stem cells through let-7f

microRNA and Wnt/b-catenin signaling. Proc Natl Acad Sci USA 109: E309–316.

72. Urban BC, Todryk S (2006) Malaria pigment paralyzes dendritic cells. J Biol 5:4.

73. Giribaldi G, Ulliers D, Schwarzer E, Roberts I, Piacibello W, et al (2004)

Hemozoin- and 4-hydroxynonenal-mediated inhibition of erythropoiesis.Possible role in malarial dyserythropoiesis and anemia. Haematologica 89:

492–493.

Haemozoin Induces Human Monocyte TIMP-1

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